Vitellogenin of the Chilean flounder Paralichthys adspersus as a biomarker of endocrine disruption along the marine coast of the South Pacific. Part I: induction, purification, and identification

Abstract

We sought to provide a useful indicator of the presence of endocrine-disrupting contaminants along the marine coast of the South Pacific using Chilean flounder (Paralichthys adspersus). In light of the lack of information on vitellogenin for this species, we induced, purified, and identified the plasma vitellogenin of Chilean flounder inhabiting the Chilean coast. Vitellogenin (Vg) from Chilean flounder was purified by size exclusion and ion-exchange chromatography using plasma from juvenile males induced by injecting 17beta-estradiol. The Vg was detected by SDS-PAGE and Western blot analyses using an antibody against turbot (Scophthalmus maximus) vitellogenin. These analyses revealed a protein band of 205 kDa and three minor bands of 120, 90, and 68 kDa. These proteins were identified as Vg by means of mass spectrometry (LCQ Duo ESI-IT-MS), matching sequences of tryptic peptides to known sequences for several other fish species. The matches showed the presence of vitellogenin (VgI, VgII, Vg A and Vg B) in Chilean flounder, similar to species such as mummichog (Fundulus heteroclitus), Japanese medaka (Oryzias latipes), and white perch (Morone americana). These results are discussed in terms of identifying Vg in Paralichthys adspersus with the antibody to turbot Vg. Moreover, we compare the molecular size of Vg from Chilean flounder (large) with that of other flatfish species. Finally, we discuss the potential use of this molecule as a biomarker for the presence of xeno-estrogenic compounds along the Chilean coastline.

Enzyme-linked immunosorbent assay (ELISA) measurement of vitellogenin in plasma and liver histopathology in barfin plaice Liopsetta pinnifasciata from Amursky Bay, Sea of Japan

“…Vitellogenin of rainbow trout under denaturing SDS-PAGE conditions also dissociated into several protein bands: one major band with a mass of 240 kDa and three minor bands with molecular masses of around 170, 90 and 30 kDa (Brion et al 2000). The major 180-kDa L. pinnifasciata fragment was comparable with that described for different flatfish species: i.e., 172 kDa for Senegalese sole Solea senegalensis (Guzmán et al 2008), 175 kDa for winter flounder Pleuronectes americanus (Hartling et al 1997) and barfin flounder Verasper moseri (Matsubara and Sawano 1995;Matsubara et al 1999), 180 (188) kDa for California halibut Paralichthys californicus (Palumbo et al 2007), 185 kDa for turbot Scophthalmus maximus (Silversand and Haux 1989) but less comparable with the major Vg fragment found for other flatfish: i.e., 205 kDa for Chilean flounder Paralichthys adspersus (Leonardi et al 2009), 160 kDa for Atlantic halibut Hippoglossus hippoglossus (Norberg 1995), 155 kDa for greenback flounder Rhombosolea tapirina (Sun et al 2003), 133 kDa for smooth flounder Pleuronectes putnami (Roy et al 2004), 130 kDa for English sole Pleuronectes vetulus (Roubal et al 1997).…”

“…Nevertheless, males do produce vitellogenin in the same quantity as females when oestrogens and oestrogen mimic substances are present in the environment. Thus, Vg detection in males is widely exploited as a biomarker of oestrogenic effects of marine environment contamination (Solé et al 2001;Vethaak et al 2002;Leonardi et al 2009). Numerous oestrogenic chemicals and waste waters have been shown to induce Vg synthesis in male fish (Jobling et al 1998;Bieberstein et al 1999;Hashimoto et al 2000;Madsen et al 2003), histopathological changes in the liver and gonads, and the time it takes to reach puberty.…”

“…Recently, ecologists have shown interest in studying fish vitellogenins (Vg) (Hashimoto et al 2000;Vethaak et al 2002;Kleinkauf et al 2004;Leonardi et al 2009). Vitellogenin is a dimeric phospholipoglycoprotein of high molecular mass, a precursor of yolk proteins that is synthesised in the female liver in response to oestrogenic stimulation.…”

“…Male fish can also synthesise measurable amounts of this protein in response to oestrogenic stimulation. Due to this ability, Vg has been proposed as a biomarker of exposure to oestrogenic chemicals in the environment Heppell et al 1995;Tyler et al 1996;Solé et al 2001;Scott et al 2006;Leonardi et al 2009). These substances can imitate the effects of natural oestrogenic hormones, such as 17b-estradiol, and adversely affect normal physiological and endocrine regulation in living organisms (Golden et al 1998).…”

Proteomic characterization of vitellogenins from three species of South American fresh water fish

“…Isoelectric point and molecular mass of the three induced proteins observed in the blood plasma of each estrogen-treated fish. , 2009Palumbo et al, 2007;Leonardi et al, 2010). In this work, 17β-estradiol (E2) was used for the induction of Vtgs synthesis in adult males of P. fasciatum, P. brachypomus and C. macropumum because this is the most common estrogen inducer of Vtgs in adult male and juvenile fish (Sundararaj & Nath, 1981;Van Bohemen et al, 1982;Mañanós et al, 1994).…”